Process for producing antibiotic baumycin complex and components

ABSTRACT

A novel anthracycline antibiotic complex designated herein as baumycin complex is produced by fermentation of a baumycin-producing strain of Streptomyces, e.g. Streptomyces coeruleorubidus ME 130-A4 (FERM-P3540, ATCC 31276). The complex and four bioactive components thereof designated baumycin A1, A2, B1 and B2 are useful as antibacterial and antitumor agents.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of our prior, copending application Ser.No. 797,201 filed May 16, 1977 and now U.S. Pat. No. 4,147,778.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to new anthracycline antibiotics, to theirproduction and recovery, and to their therapeutic use.

2. Description of the Prior Art

A number of anthracycline glycosides have been described in theliterature. Among them, daunomycin and adriamycin are particularly beingwatched with keen interest in the field of cancer chemotherapy and havealready been applied clinically for human cancers. Preparation ofadriamycin by fermentation of S. peuceticus var. caesius is disclosed inU.S. Pat. No. 3,590,028. Chemical conversion of daunomycin to adriamycinis taught in U.S. Pat. No. 3,803,124. Daunomycin (produced byfermentation of S. peuticus in U.K. Pat. No. 1,003,383) may be the sameas Rhone-Poulenc's 13,057 R.P. (formerly rubidomycin and nowdaunoribicin: see U.K. Pat. No. 985,598, 1,188,262 and 1,241,750 andU.S. Pat. No 3,616,242) and is probably identical to Ciba's danubomycindisclosed in U.S. Pat. No. 3,092,550 and U.K. Pat. No. 901,830. See alsoU.S. Pat. No. 3,686,163 on dihydrodaunomycin. Cinerubin A and CinerubinB, glycosides having ε-pyrromycinone, are disclosed in U.K. Pat. No.846,130 [see also U.S. Pat. No. 3,864,480 and Keller-Schierlein, et.al., Antimicrobial Agents and Chemotherapy, page 68 (1970 ) and ChemicalAbstracts, 54, 1466i (1960)]. The anthracycline glycoside carminomycindescribed in J. Antibiotics 27:254-259 (1974), in West GermanSpecification No. 2,362,707 and in J. Amer. Chem. Soc. 97 (20):5955-5956(1975) has been reported to be active in several animal tumor systems.The antibiotic pyrromycin disclosed in Chem. Ber. 92:1904-1909 (1959)contains an aglycone ε-pyrromycinone and an amino sugar rhodosamine. Forfurther illustrative and summary disclosures of anthracyclineantibiotics see Index of Antibiotics from Actinomycetes, Hamao Umezawa,Editor-in-Chief, University Park Press, State College, PA, U.S.A. (1967)as follows:

    ______________________________________                                        Antibiotic           Page Number                                              ______________________________________                                        Aklavin              111                                                      Cinerubin A          220                                                      Cinerubin B          221                                                      Danubomycin          242                                                      Daunomycin           243                                                      Pyrromycin           524                                                      Rhodomycin A, B      561                                                      Rubidomycin          574                                                      ______________________________________                                    

The textbook Antibiotics, Vol. 1, Mechanism of Action, edited by DavidGottlieb and Paul D. Shaw, Springer--Verlag New York, Inc., N.Y., N.Y.(1967) at pages 190-210 contains a review by A. DiMarco entitledDaunomycin and Related Antibiotics. Information Bulletin, No. 10,International Center of Information of Antibiotics, in collaborationwith WHO, December 1972, Belgium, reviews anthracyclines and theirderivatives.

SUMMARY OF THE INVENTION

This invention relates to new anthracycline glycoside antibiotics, toprocesses for their preparation, to pharmaceutical compositionscontaining them and to the use of such antibiotics or compositions inthe treatment of bacterial infections and in the inhibition of mammaliantumors. More particularly, it relates to a novel anthracycline glycosidecomplex designated herein as baumycin complex and to the individualbioactive components of said complex designated baumycin A₁, A₂, B₁ andB₂. The complex and above-mentioned components are produced bycultivating a baumycin-producing strain of Streptomyces, most preferablyStreptomyces coeruleorubidus ME 130-A4 (FERM-P3540, ATCC 31276), in anaqueous nutrient medium containing assimilable sources of carbon,nitrogen, inorganic salts and other factors necessary for the growth ofthe microorganism under submerged aerobic conditions until a substantialamount of baumycin is produced by said microorganism in said culturemedium and, optionally, recovering the baumycin from the culture medium.Baumycin A₁, A₂, B₁ and B₂ may be recovered and separated by extractionof the whole fermentation broth, with or without the separation ofmycelium, or by extraction from mycelium followed by separation andisolation of the component antibiotics by standard columnchromatographic procedures. Baumycin complex and its componentanthracycline glycosides, i.e. baumycin A₁, A₂, B₁ and B₂, inhibit thegrowth of gram-positive bacteria and inhibit the growth of variousmammalian tumors.

This invention also embraces baumycin complex and its components,baumycin A₁, A₂, B₁ and B₂, as crude solids, as purified solids, astheir non-toxic acid addition salts and as complexes withdeoxyribonucleic acid.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the ultraviolet and visible light absorption spectrum ofbaumycin A₁ in methanol.

FIG. 2 shows the infrared absorption spectrum of baumycin A₁ whenpelleted in potassium bromide.

FIG. 3 shows the NMR spectrum of baumycin A₁ in CDCl₃ (100 MHz).

FIG. 4 shows the ultraviolet and visible light absorption spectrum ofbaumycin A₂ in methanol.

FIG. 5 shows the infrared absorption spectrum of baumycin A₂ whenpelleted in potassium bromide.

FIG. 6 shows the NMR spectrum of baumycin A₂ in CDCl₃ (100 MHz).

FIG. 7 shows the ultraviolet and visible light absorption spectrum ofbaumycin B₁ in methanol.

FIG. 8 shows the infrared absorption spectrum of baumycin B₁ whenpelleted in potassium bromide.

FIG. 9 shows the NMR spectrum of baumycin B₁ in a mixture of CDCl₃ andCD₃ OD (100 MHz).

FIG. 10 shows the ultraviolet and visible light absorption spectrum ofbaumycin B₂ in methanol.

FIG. 11 shows the infrared absorption spectrum of baumycin B₂ whenpelleted in potassium bromide.

FIG. 12 shows the NMR spectrum of baumycin B₂ in a mixture of CDCl₃ andCD₃ OD (100 MHz).

FIG. 13 shows the field desorption mass spectrum of baumycin A₁ (emittercurrent: 11 mA).

FIG. 14 shows the field desorption mass spectrum of baumycin A₂ (emittercurrent: 12 mA).

FIG. 15 shows the field desorption mass spectrum of baumycin B₁ methylester derivative (emitter current: 14 mA).

FIG. 16 shows the field desorption mass spectrum of baumycin B₂ methylester derivative (emitter current: 14 mA).

DETAILED DESCRIPTION

The present invention provides the novel anthracycline glycosides of thegeneral formula ##STR1## wherein R represents ##STR2## and the non-toxicacid addition salts and complexes thereof with deoxyribonucleic acid.

The compounds of formula I are components of a fermentation-producedanthracycline complex designated herein as baumycin complex. Baumycincomplex thus comprises the anthracycline glycosides of the formula##STR3## designated baumycin A and the anthracycline glycosides of theformula ##STR4## designated baumycin B. It has been established thatbaumycin A and B can each be separated into two bioactive stereoisomersand thus, formula IA above represents the isomers baumycin A₁ and A₂while formula IB represents baumycin B₁ and B₂. As used herein the termbaumycin means the antibiotic comprising at least one of baumycin A₁,A₂, B₁ and B₂.

THE MICROORGANISM

The compounds of the present invention are produced by fermentation ofvarious baumycin-producing strains of Streptomyces including severalknown daunomycin-, adriamycin- and carminomycin- producing strains suchas Streptomyces peuceticus subsp. carneus ATCC 21354, Streptomycescoeruleorubidus ATCC 13740, Streptomyces peuceticus subsp. caestus NRRLB-5337, Streptomyces peuceticus NRRL B-3826 and Streptomycescoeruleorubidus NRRL B-3045.

An especially preferred baumycin-producing strain has been isolated bythe present inventors from a soil sample collected at the Institute ofMicrobial Chemistry, Osaki, Tokyo, Japan and designated strain ME130-A4. Cultures of this strain have been deposited in the American TypeCulture Collection, Rockville, Maryland and in the Fermentation ResearchInstitute, Japan, and added to their permanent collections ofmicroorganisms as ATCC 31276 and FERM P-3540 respectively.

Strain ME 130-A4 has the following properties:

(1) Morphological properties:

Under microscope, open spirals and hooks are observed to develop wellfrom branched substrate mycelia. No whorls, and mature spore chain ismoderately long with more than ten spores. The spores measure 0.6 to0.8μ×1.0 to 1.2μ and their surface is spiny.

(2) Growth on various media:

The description in parenthesis follows the color standard "Color HarmonyManual" published by Container Corporation of America, U.S.A.

(a) On glycerol-asparagine agar (ISP medium No. 5), incubated at 27° C.:Pale reddish yellow to dark red (4ic, Pastel orange to 61/2 nc, Catchup)growth; light gray (17 ge, Dusty Aqua Blue to 19 fe, Aqua Gray) aerialmycelium; light reddish yellow soluble pigment.

(b) On sucrose-nitrate agar, incubated at 27° C.: Pale reddish yellow,pale red to dark red (61/2 le, Cedar) growth; slight white aerialmycelium; slight dark red soluble pigment.

(c) On glucose-asparagine agar, incubated at 27° C.: Colorless, paleyellow to pale orange growth; slight white to light gray aerial myceliumthen becoming abundant after 14 days incubation; no soluble pigment.

(d) On starch-inorganic salts agar (ISP medium No. 4), incubated at 27°C.: Pale yellowish brown to pale reddish yellow growth; light gray (19dc, Aqua Gray) aerial mycelium; no soluble pigment.

(e) On tyrosine agar (ISP medium No. 7), incubated at 27° C.: Grayishred brown to brown (4 ni, Chestnut Brown) growth; blue white to lightblue (19 dc, Aqua Gray) aerial mycelium; dark brown soluble pigment.

(f) On nutrient agar, incubated at 27° C.: Yellowish brown growth; whiteaerial mycelium; brown soluble pigment.

(g) On yeast extract-malt extract agar (ISP medium No. 2), incubated at27° C.: Dull orange (5 ne, Tile Red) growth; light gray (19 fe, AquaGray) aerial mycelium; slight brown soluble pigment.

(h) On oatmeal agar (ISP medium No. 3), incubated at 27° C.: Colorlessto pale orange growth; light gray (19 fe, Aqua Gray) aerial mycelium; nosoluble pigment.

(i) On glycerol-nitrate agar, incubated at 27° C.: Colorless to paleorange growth; white to light gray aerial mycelium; no soluble pigment.

(j) On starch agar, incubated at 27° C.: Light orange growth; white tolight gray aerial mycelium, which is faint for 14 days incubation;slight pale orange soluble pigment.

(k) On calcium malate agar, incubated at 27° C.: Colorless, pale orangeto dull orange growth, white to light gray (17 ge, Dusty Aqua Blue)aerial mycelium; slight pink soluble pigment.

(l) On cellulose, incubated at 27° C.: Colorless growth; white to dullblue green aerial mycelium; no aerial mycelium.

(m) On gelatin stab, incubated in 20° C.: Slight yellowish brown growth;no aerial mycelium, slight brown soluble pigment.

(n) On glucose-peptone-gelatin stab, incubated at 27° C.: Pale yellowishbrown to yellowish brown growth; slight white aerial mycelium; darkbrown soluble pigment.

(o) On skimmed milk, incubated at 27° C.: Pale yellowish brown,yellowish brown to pale red growth; slight white aerial mycelium; brownsoluble pigment.

(3) Physiological properties:

(a) Growth temperature was examined on glucose-asparagine agar at 20,24, 27, 30, 37 and 50° C. and optimal temperature is about 30° to 37° C.

(b) Gelatin liquefaction on 15% gelatin stab at 20° C. and onglucose-peptone-gelatin stab at 27° C.: On the former medium, gelatinliquefaction was observed weakly after 20 days incubation, but on thelatter liquefaction began weakly or moderately after 14 days incubation.

(c) Starch hydrolysis on starch-inorganic salts agar and on starch agarat 27° C.: Strong hydrolysis was observed after 3 days incubation on theformer medium and after 5 days incubation on the latter medium.

(d) Peptonization and coagulation of skimmed milk at 37° C.: Weak tomoderate peptonization began after 7 days incubation and finishedcoagulation on around 10 to 14 days.

(e) Melanin formation on tryptone-yeast extract broth (ISP medium No.1), on peptone-yeast extract ferrous agar (ISP medium No. 6) and ontyrosine agar (ISP medium No. 7) at 27° C.: Positive on all media.

(f) Utilization of carbohydrates of Pridham-Gottlieb basal medium (ISPmedium No. 9), incubated at 27° C.: Abundant growth with glucose,L-arabinose, D-xylose, sucrose, rattinose.

(g) Liquefaction of calcium malate on calcium malate agar at 27° C.:Strong to moderate liquefaction around the growth was observed after 3days incubation.

(h) Nitrate reduction on peptone water containing 1% sodium nitrate (ISPmedium No. 9), incubated at 27° C.: Negative.

Summarizing the above characteristics of No. ME 130-A4, the strainbelongs to the genus Streptomyces. Aerial mycelium forms open spirals,but no whorls. The spore surface is spiny. The growth on various mediais found to be pale orange to dull red and aerial mycelium is lightgray. Slight pale orange soluble pigment is produced. Melanin formationis positive. Proteolytic action is weak to moderate and starchhydrolysis is strong. Among known species of Streptomyces, strain No. ME130-A4 resembles Actinomyces coeruleorubidus based on theabove-mentioned properties. (Reference 1: International Journal ofSystematic Bacteriology, 18, 312, 1968, Ref. 2: G. F. Gause, ZurKlassifizierung der Actinomyceten, p. 98 Veb. Guotab Fischer VerlagJena, 1958).

Strain ME 130-A4 and Act. coeruleobidus ISP 5145 were compared byparallel cultures. The results are as follows:

    ______________________________________                                                               Act. coeruleorubidus                                                 ME 130-A4                                                                              ISP 5145                                               ______________________________________                                        Spirals         positive   positive                                           Spore surface   spiny      spiny                                              Aerial mycelium light gray light gray                                         Growth          pale orange                                                                              pale yellowish                                                     dull red   brown to dull red                                  Melanin formation                                                              ISP medium No. 1                                                                             positive   positive                                            ISP medium No. 6                                                                             "          "                                                   ISP medium No. 7                                                                             "          "                                                  Starch hydrolysis                                                                             "          "                                                  Coagulation of milk                                                                           "          "                                                  Peptonization of milk                                                                         "          "                                                  Liquefaction of gelatin                                                                       "          "                                                  Nitrate reduction                                                                             negative   negative                                           Utilization of carbohydrates                                                   Glucose        positive   positive                                            L-Arabinose    "          "                                                   D-Xylose       "          "                                                   D-Fructose     "          "                                                   Sucrose        "          "                                                   Inositol       "          "                                                   L-Rhamnose     "          "                                                   Raffinose      "          "                                                   D-Mannitol     "          "                                                  Optimal temperature                                                                           around     around                                             for growth      37° C.                                                                            37° C.                                      Antibiotics produced                                                                          Baumycin   Rubidomycin                                                        Daunomycin (Daunomycin)*                                      ______________________________________                                         *Reference, Journal of Pharmaceutical Science 56, 1691 p, 1967.          

From the results, it can be seen that the present strain ME 130-A4agrees very closely with Actinomyces coeruleorubidus in morphologicaland physiological properties. There is only a little difference in thecolor of the growth, in which the ME 130-A4 strain is a little more redthan Act. coeruleorubidus.

According to reference 2, nitrate reduction by St. coeruleorubidus isvariable depending upon the strain of this species. Thus, strain No. ME130-A4 can be identified as a new strain of Streptomycescoeruleorubidus.

Since the Streptomyces are easily mutatable naturally or artificially,S. coeruleorubidus No. ME 130-A4 and the other baumycin-producingStreptomyces of the present invention include the typical strainsdescribed above and all natural and artificial baumycin-producingvariants and mutants thereof.

PRODUCTION OF BAUMYCIN

Production of the compounds of the present invention is carried out bycultivating a baumycin-producing strain of Streptomyces in aconventional aqueous nutrient medium containing known nutritionalsources for actinomycetes, i.e. sources of carbon, nitrogen andinorganic salts. Submerged aerobic culture is preferably employed forthe production of substantial amounts of baumycin, just as for otherantibiotics. The general procedures used for the cultivation of otheractinomycetes are applicable to the cultivation according to thisinvention. The medium preferably contains commercially available carbonsources such as glycerol, glucose, starch, dextrin, sucrose, maltose,oils, fats and the like in either purified or crude state andcommercially available nitrogen sources such as soybean powder, yeastextract, peptone, cotton seed powder, dried yeast, corn steep liquor orinorganic salts such as ammonium sulfate, sodium nitrate or ammoniumchloride. Inorganic salts such as sodium chloride, potassium chloride orphosphates are preferably used and there may also be added, ifnecessary, trace metals and defoamers such as Adekanol (Trademark, AsahiDenka Ind. Co.) or silicone (Trade mark, Shinetsu Chem. Ind. Co.). Theculture temperature should be in the range of about 20°-35° C.,preferably about 25°-30° C. Production of baumycin in the culture brothreaches a maximum after 3 to 7 days in either shake flask or submergedaerobic fermentation with aeration and agitation provided as in theexamples shown below.

SEPARATION AND ISOLATION OF BAUMYCIN COMPONENTS

The compounds, baumycin, in the present invention can be recovered fromthe culture broth and separated from each other by the followingprocedures.

Baumycin produced by fermentation exists intracellularly as well asextracellularly, but is found mainly in the mycelium. To recoverbaumycin complex from the culture broth, the broth may be filtered andthe filtrate then extracted with a water-immiscible organic solvent suchas ethyl acetate, butyl acetate, chloroform or n-butanol. Baumycin inthe mycelium can be recovered by extraction with an organic solvent suchas chloroform, acetone, n-butanol, methanol, ethanol, ethyl acetate ormethyl ethyl ketone or an aqueous solution of an organic or inorganicacid such as hydrochloric acid, sulfuric acid or acetic acid.Alternatively, baumycin can be extracted directly from the culture brothby the above-mentioned extraction procedures without prior separation ofthe mycelium. After concentrating in vacuo, the baumycin extracts may bere-extracted with a water-immiscible organic solvent at a pH between 7and 9 and the baumycin then dissolved into an acidic aqueous solutionhaving a pH<4. Baumycin in said acidic aqueous solution is thenre-extracted with an organic solvent after adjustment to a weakly basicpH. By repeating the above procedures as necessary, baumycin complex canbe prepared in a purified form. As an alternative to using a solventextraction recovery method of in combination with such a method,baumycin may be recovered from the culture broth by columnchromatography using adsorbents such as activated carbon, alumina,silicic acid, or a modified dextran such as that commercially availableunder the trade name Sephadex LH-20 (Pharmacia Fine Chemical Co., NewYork, N.Y.), countercurrent distribution or liquid chromatography usingsuitable organic solvents. Active extracts obtained by such methods areconcentrated under reduced pressure and obtained as the crude red powderof baumycin complex.

To obtain the individual baumycin components A₁, A₂, B₁ and B₂ from thebaumycin complex, further purification and separation may be carried outusing such standard separation techniques as column chromatography usingvarious adsorbents such as silicic acid, modified dextrans (e.g.Sephadex LH-20), weakly acidic ion-exchange resins or activated carbon,countercurrent distribution or liquid chromatography using suitableorganic solvents, or a combination of one or more of the above-mentionedprocesses. As an example of a suitable separation procedure, baumycincomplex may be dissolved in a small amount of chloroform, subjected tocolumn chromatography over silicic acid and then eluted with a suitableorganic solvent, e.g. chloroform-methanol, to give baumycin componentsA₁, A₂, B₁ and B₂. The active eluates are separated, concentrated underreduced pressure and the baumycin components individually purified bychromatography over Sephadex LH-20. After concentration of the activeeluates, baumycin A₁, A₂, B₁ and B₂ may be obtained in purifiedcrystalline form by recrystallization from a suitable organic solvent.

PHYSICOCHEMICAL PROPERTIES OF BAUMYCIN COMPONENTS

The physicochemical properties of baumycin A₁, A₂, B₁ and B₂ are asfollows:______________________________________Baumycin A₁A₂______________________________________Appearance Weakly basisamorphous red powderElementaryanalysis C H N O C H NO______________________________________Found 59.85 6.65 2.04 29.83 60.276.72 2.31 29.52Calcd. 60.61 6.43 2.08 30.88 60.61 6.43 2.0830.88Empirical C₃₄ H₄₃ NO₁₃formulaMolecular 673.7weightMelting point (°C.) 182-185 185-189Specificrotation + 150° + 135° [α]_(D) ²⁰ (CHCl₃, C =0.1) Soluble in acidic water, DMSO, methylcello- solve, methanol,ethanol, n-propanol, n-butanol ethyl acetate, butyl acetate,Solubilityacetone, methyl ethyl ketone, methylene chloride and chloroform.Insoluble in water, n-hexane, cyclohexane and petroleum ether.R_(f)values ***C:M = 10:1 0.25 0.08C:M:B =7:3:3 0.39 0.28C:M:F =90:10:1 0.260.17C:M:A =80:20:4 0.74 0.64 Acidic aqueous and methanol solution is redand turns to reddish purple in alkaline state.Reaction Baumycin A₁ andA₂ give positive ninhydrin reaction, and do not reduce Fehlingsolution.______________________________________UVand visible FIG. 1 FIG.4absorptionspec- 234.5 (452), 252.5 (327), 234.5 (427), 252.5 (311),traand max 289 (120), 478 (127), 289 (108), 478 (125),1% 497 (128), 532(76) 497 (128), 532 (84)E_(1cm))in MeOH(curve 1)in 0.1N HCl- 234.5(459), 252.5 (326), 234.5 (447), 252.5 (311),MeOH 289 (121), 479 (133),289 (111), 478 (131),(curve 2) 497 (133), 532 (78) 497 (131), 532 (70)in0.1N 250.5 (416), 350 (83), 250.5 (421), 350 (80),NaOH-MeOH 558 (164),597 (152) 558 (170), 596 (164)(curve 3)Infraredabsorp-tion spectrum FIG.2 FIG. 5 (KBr)NMRspectrum FIG. 3 FIG. 6 (PMR) (100 MHz, in CDCl₃)***C¹³NMR Characteristic Characteristicspectrum C-1" peak C-1" peak at 106.7ppm at 101.6 ppm______________________________________ *C = chloroform,M = methanol, B = benzene, F = formic acid, A = acetic acid **TLCcondition: silicic acid thin layer 60F₂₅₄ (Merck Co.), 23° C.***Spectrum measured on Varian XL 100 instrument at 25.2 MHz. Internalreference is CDCl₃ for baumycin A₁ and TMS for baumycin A₂ Samples: A₁ =27 mg./0.5 ml. CDCl₃ ; A₂ =44 mg./0.6 ml.CDCl₃.______________________________________Baumycin B₁B₂______________________________________Appearance Weakly basicamorphous red powderElementaryanalysis C H N O C H NO______________________________________Found 57.32 6.45 2.01 32.58 56.595.96 1.92 31.91Calcd. 59.38 6.01 2.04 32.57 59.38 6.01 2.0432.57Empiricalformula C₃₄ H₄₁ NO₁₄Molecularweight 687.7Melting point (°C.) 181-189 197-201Specificrotation + 170° + 170° [α]_(D) ²⁰ (CHCl₃ :MeOH = 1 : 1, C = 0.1) Soluble in acidic water, methyl cellosolve,methanol, ethanol, n-propanol and n-butanol. Insoluble in water, ethylacetate, acetone,Solubility methylene chloride, chloroform, carbontetra- chloride, benzene, toluene, ethyl ether and n-hexane, except thatbaumycin B₂ is soluble in water.R_(f) values***C:M = 10:1 0.07 0.01C:M:B=7:3:3 0.39 0.14C:M:F =90:10:1 0.18 0.10C:M:A =80:20:4 0.64 0.30 Acidicaqueous and methanol solution is red and turns to reddish purple inalkaline state.Reaction Baumycin B₁ and B₂ give positive ninhydrinreaction, and do not reduce Fehling solution.UVand visible FIG. 7 FIG.10absorptionspec- 234.5 (552), 253 (385), 234 (575), 252 (414),tra andmax 290 (132), 476 (179), 290 (130), 478 (176),(E^(1%) _(1cm)) 495(181), 530 (101) 495 (183), 530 (120)in MeOH(curve 1)in 0.1N HCl- 234(580), 253 (397), 234 (616), 253 (419),MeOH 290 (140), 476 (182), 290(145), 476 (195),(curve 2) 495 (184), 530 (100) 494 (191), 529 (105)in0.1N 251 (453), 350 (65), 251 (499), 350 (74),NaOH-MeOH 556 (206), 594(195) 556 (231), 594 (218)(curve 3)Infraredabsorp-tion spectrum FIG. 8FIG. 11(KBr)NMRspectrum FIG. 9 FIG. 12(PMR) (100 MHz, in CDCl₃ and CD₃OD mixture)***C¹³ NMR Characteristic Characteristicspectrum C-1" peakC-1" peak at 107.1 ppm at 102.1ppm______________________________________ *C = chloroform, M = methanol,B = benzene, F = formic acid, A = acetic acid **TLC condition: silicicacid thin layer 60F₂₅₄ (Merck Co.), 23° C. ***Spectrum measured onVarian XL100 instrument at 25.2 MHz. Internal reference is TMS. Samples:B₁ = 45 mg./0.6 ml. CDCl₃ :methanol (5:1); B₂ = 30 mg./0.6 ml. CDCl₃:methanol (1:1).

STRUCTURE DETERMINATION

The structures of baumycin A₁, A₂, B₁ and B₂ in the present inventionwere determined as follows: On hydrolysis with 0.1 N hydrochloric acidfor 30 min. at 85° C., baumycin A₁, A₂, B₁ and B₂ give daunomycinone anddaunosamine, and daunomycin is obtained from the above baumycincomponents on partial hydrolysis with 1% sulfuric acid for 15 min. at32° C. Physicochemical properties such as NMR, mass and infra-redabsorption spectra, melting point and R_(f) values on thin-layer ofdaunomycinone, daunosamine and daunomycin obtained from baumycin A and Bby acid hydrolysis coincided fully with those of authentic daunomycin(Journal of American Chemical Society, 86, 5334-5335, 5335-5336 (1964)).

Further elucidation of the structures of baumycin A₁ and A₂ in thepresent invention was carried out as follows: The hydrogenolysis ofbaumycin A₂ with Pd/BaSO₄ in methanol gave an aglycone portion and asugar moiety. From analysis of PMR and mass spectral data, the aglyconewas determined to be 7-deoxydaunomycinone. With regard to the sugarmoiety of baumycin A₁ and A₂, the tetracetyl derivative, which isobtained by the treatment of either moiety (i.e. that of A₁ or A₂) withacetic anhydride in pyridine, was analyzed by PMR and CI (chemicalionization) mass spectra and the following structure proposed: ##STR5##To further confirm the structures of baumycin A₁ and A₂, the molecularion peak of each was determined by the recently developed FD (fielddesorption) mass spectrum analysis and was found to be m/e=674 (M+1) asshown in FIG. 13 and FIG. 14. Accordingly, the molecular formula forboth baumycin A₁ and A₂ is C₃₄ H₄₃ NO₁₃. In view of the differences inmelting points, specific rotations, thin layer chromatography R_(f)values and C¹³ NMR peaks, baumycin A₁ and A₂ have been determined to bestereoisomers of each other.

The structures of baumycin B₁ and B₂ were determined as follows:Hydrogenolysis of baumycin B₁ with Pd/BaSO₄ in methanol gave an aglyconeportion and a sugar moiety. From analysis of the PMR and mass spectraldata, the aglycone portion was determined to be 7-deoxydaunomycinone.With regard to the sugar moiety, the diacetyl derivative thereof, whichis obtained by treatment of each sugar moiety with acetic anhydride inpyridine, was analyzed by PMR and CI mass spectra and the followingstructure proposed: ##STR6## To further confirm the structures ofbaumycin B₁ and B₂, the molecular ion peak was determined for each by FDmass spectrum. Since the ion peak could not be obtained from baumycin B₁and B₂ per se, their methyl ester derivative obtained by treatment withdiazomethane was analyzed and showed a molecular ion peak at m/e=702(M+1) as indicated in FIG. 15 and FIG. 16. The peaks at m/e=716, 730 and744 shown in FIG.'s 15 and 16 indicate the methylation of the aminoresidues in daunosamine. While baumycin B₁ and B₂ thus have the samemolecular formula, it has been established from differences in theirmelting points, thin layer chromatography R_(f) values and C¹³ NMR peaksthat they are stereoisomers of each other.

Summarizing the results of the above work, baumycin A₁ and A₂ have thegeneral structure ##STR7## while baumycin B₁ and B₂ have the generalstructure ##STR8## As mentioned above baumycins A₁ and A₂ and B₁ and B₂are stereoisomers of each other and can be easily distinguished bydifferences in such physical properties as melting points, specificrotation (A₁ and A₂), thin layer R_(f) values and C¹³ NMR peaks.

It will be readily seen from the above structures that baumycin A₁, A₂,B₁ and B₂ are novel anthracycline glycoside antibiotics which containthe same aglycone and the same amino sugar (i.e. daunosamine) asdaunomycin but which differ from daunomycin in having the additionalnovel sugar moieties shown above. The baumycin antibiotics of thepresent invention have also been differentiated by the present inventorsfrom known anthracycline antibiotics by R_(f) value comparisons usingsilica gel thin layer chromatography with various solvent systems.

ANTIBIOTIC ACTIVITY OF BAUMYCIN

Baumycin A₁, A₂, B₁ and B₂ exhibit antimicrobial activities againstvarious kinds of microorganisms. The minimum inhibitory concentration ofthe present antibiotics as determined by the broth dilution method areshown in Table 1.

                  Table 1                                                         ______________________________________                                        Antimicrobial spectrum of baumycin A.sub.1, A.sub.2, B.sub.1 and B.sub.2                    Minimun inhibitory                                                            concentration (mcg./ml)                                         Test organism   BM-A.sub.1                                                                             BM-A.sub.2                                                                            BM-B.sub.1                                                                          BM-B.sub.2                             ______________________________________                                        Staph. aureus FDA209P                                                                         1.56     3.12    50     100                                   Staph. aureus Smith                                                                           0.78     1.56    50      50                                   B. subtilis ATCC 6633                                                                         0.78     3.12    100   >100                                   B. cereus ATCC 9634                                                                           1.56     3.12    100   >100                                   B. megaterium NRRL B-938                                                                      1.56     6.25    100   >100                                   Sarcina lutea ATCC 9341                                                                       0.78     3.12    50      25                                   Micrococcus flavs                                                                             0.78     1.56    50      50                                   Coryne. bovis 1810                                                                            1.56     6.25    50      50                                   Ps. fluorescens                                                                NIHJB-254      >100     100     100   >100                                   Proteus morganii                                                                              >100     >100    >100  >100                                   Mycobacterium                                                                 smegmatis ATCC 607                                                                            6.25     12.5    100   >100                                   Candida albicans                                                               IAM 4905       100      100     100   >100                                   Candida tropicalis                                                                            100      100     100   >100                                   ______________________________________                                         BM: baumycin                                                             

As shown above, baumycin A₁, A₂, B₁ and B₂ in the present inventionpossess antimicrobial activity, especially against gram-positivebacteria, and thus they are therapeutically useful in the treatment ofanimals, including man, for diphtheria, tuberculosis, pneumonia, tetanusand other infectious diseases caused by gram-positive bacteria.

ANTITUMOR ACTIVITY OF BAUMYCIN

Baumycin A₁, A₂, B₁ and B₂ in the present invention show a markedantitumor activity with low toxicity in experimental animal tests andthus are therapeutically useful in inhibiting the growth of mammaliantumors. In particular, baumycin A₁, A₂, B₁ and B₂ showed markedinhibitory effects on mouse L-1210 leukemia. For example, BDF₁ mice wereinoculated intraperitoneally with 1×10⁶ L-1210 cells/mouse and 24 hrs.after inoculation the drug was intraperitoneally injected once daily for10 days consecutively. On day 30, the % of prolongation of the survivaltime to control was as follows:______________________________________Prolongation of the survival timeDose T/C (%)(mg./kg./day BM-A₁ BM-A₂BM-B₁ BM-B₂______________________________________8 147 1556 165 1354 1361112 117 991 167 105 990.5 173 990.25 163 990.125 151 930.06 >300141____________________________________________________________________________Prolongation of the survival timeDose T/C (%)(mg./kg./day) BM-A₁ BM-A₂BM-B₁ BM-B₂______________________________________0.03 1870.015 1550.0081310.004 131______________________________________

ACUTE TOXICITY

The LD₅₀ values upon intraperitoneal injection of the antibiotics of thepresent invention are shown in the followingtable.______________________________________ LD₅₀(mg./kg.)______________________________________BM-A₁ 1.5-2.5BM-A₂15-20BM-B₁ 40-60BM-B₂ 75-100______________________________________ BM:baumycin

THE THERAPEUTIC USE OF BAUMYCIN

As mentioned above, the compounds baumycin A₁, A₂, B₁ and B₂ in thepresent invention are novel antibiotics, useful in both human andveterinary medicine, and also possess marked inhibitory action againstmammalian malignant tumors, especially ascitic and solid tumors.

The compounds in the present invention form non-toxic acid additionsalts with a variety of organic and inorganic salt-forming reagents andform non-toxic complexes with deoxyribonucleic acid. Thus, acid additionsalts formed with such pharmaceutically acceptable acids as sulfuric,phosphoric, hydrochloric, acetic, propionic, oleic, palmitic, citric,succinic, tartaric, glutamic, pantothenic, etc. and non-toxic complexeswith deoxyribonucleic acid can be employed in the same manner as thebaumycin compounds per se. The salts are formed, isolated, purified andformulated by the methods generally employed in salt formation forantibiotics. In the case of the DNA complexes, DNA extracted fromanimals and microorganisms such as calf thymus, Hela cells, human andanimal embryonic cells, yeasts, etc. can be used. Preparation ofbaumycin-DNA complexes can be carried out by methods described in theliterature for preparing DNA complexes of other anthracyclineantibiotics such as adriamycin, daunorubicin, etc. [see, for example,Nature, New Biol. 239:110 (1973) and Europ. J. Cancer 10:399(1974)]. Forpurposes of this invention, the baumycin compounds in the free base formare equivalent to their non-toxic acid addition salts and DNA-complexes.

According to another aspect of this invention, a method is provided fortherapeutically treating a mammalian host affected by a gram-positivebacterial infection or by a malignant tumor (e.g. a solid orascitic-type tumor such as L-1210 leukemia) which comprisesadministering to said host an effective antibacterial ortumor-inhibiting dose of baumycin A₁, A₂, B₁ and B₂, or a mixturethereof, or a non-toxic acid addition salt or DNA-complex thereof.

According to another aspect of this invention, a pharmaceuticalcomposition is provided which comprises an effective antibacterial ortumor-inhibiting amount of baumycin A₁, A₂, B₁ or B₂, or a mixturethereof, or a non-toxic acid addition salt or DNA-complex thereof, incombination with an inert pharmaceutically acceptable carrier ordiluent. These compositions may be made up in any pharmaceutical formappropriate for parenteral administration.

Preparations according to the invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions oremulsions. They may also be manufactured in the form of sterile solidcompositions which can be dissolved in sterile water, physiologicalsaline or some other sterile injectable medium immediately before use.

It will be appreciated that the actual preferred amounts of the baumycinantibiotic used will vary according to the particular compound beingused, the particular composition formulated, the mode of application andthe particular situs, host and disease being treated. In general thebaumycin antibiotics are injected intraperitoneally, intravenously,subcutaneously or locally into animals and intravenously or locally intohumans. Many factors that modify the action of the drug will be takeninto account by those skilled in the art, for example, age, body weight,sex, diet, time of administration, route of administration, rate ofexcretion, condition of the patient, drug combinations, reactionsensitivities and severity of the disease. Administration can be carriedout continuously or periodically within the maximum tolerated dose.Optimal application rates for a given set of conditions can beascertained by those skilled in the art using conventional dosagedetermination tests in view of the above guidelines.

For use as an antibacterial agent, the baumycin compositions are ingeneral administered so that the concentration of active ingredient isgreater than the minimum inhibitory concentration for the particularorganism being treated.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLE 1

A nutrient medium having the following composition was prepared:

    ______________________________________                                        Potato starch         1%                                                      Glucose               1%                                                      "Prorich" (Soybean powder)                                                                          1.5%                                                    K.sub.2 HPO.sub.4     0.1%                                                    MgSO.sub.4 . 7H.sub.2 O                                                                             0.1%                                                    NaCl                  0.3%                                                    Mineral*              0.125% (pH 7.4)                                         ______________________________________                                        *Mineral    CuSO.sub.4 . 5H.sub.2 O                                                                   2.8 g                                                             FeSO.sub.4 . 7H.sub.2 O                                                                   0.4 g                                                             MnCl.sub.2 . 4H.sub.2 O                                                                   3.2 g                                                             ZnSO.sub.4 . 7H.sub.2 O                                                                   0.8 g                                                           in 500 ml of water                                                  ______________________________________                                    

Fifty ml. of this medium was sterilized at 120° C. for 15 min. in a 500ml.--flask which was inoculated from an agar slant culture ofSteptomyces coeruleorubidus ME 130-A4 by platinum loop.

Incubation proceeded for 72 hrs. at 28° C. on a rotary shaker (230 rpm).This is the seed culture, Seven liters of the following medium wasprepared, and 50 ml. of the medium distributed and sterilized in a 500ml.--flask was aseptically inoculated with 1 ml. of the above seedculture. Fermentation was carried out at 28° C. for 7 days on a rotaryshaker (230 rpm).

    ______________________________________                                        Sucrose             4          %                                              "Prorich"           2.5        "                                              (Soybean protein, Ajinomoto                                                   Co.)                                                                          NaCl                0.25       "                                              Calcium carbonate   0.32       "                                              Mineral*            0.125      "(pH 7.4)                                      ______________________________________                                        *Mineral    CuSO.sub.4 . 5H.sub.2 O                                                                   1.25       g                                                      MnCl.sub.2 . 4H.sub.2 O                                                                   1.25       "                                                      ZnSO.sub.4 . 7H.sub.2 O                                                                   12.5       "                                                    in 500 ml. of water                                                 ______________________________________                                    

The cultured broth obtained was filtered to separate the culturefiltrate and mycelium. The filtrate was extracted three times with 1/5volume of chloroform. The mycelium was extracted three times with 2 l ofacetone per 1 kg. of cake, and the resulting acetone extract wasconcentrated to half volume under reduced pressure.

The concentrate was extracted three times with 2 l of chloroform,combined with the chloroform solution which was obtained from theculture filtrate, and concentrated to dryness under reduced pressure.Ten g. of oil substance obtained was dissolved in 50 ml. of chloroform,and the precipitate formed by addition of 300 ml. of n-hexane wascentrifuged for 5 min. at 3000 rpm to remove impure substances insolublein n-hexane. The resulting precipitate (1.4 g.) was dissolved in 100 ml.of chloroform and extracted three times with 150 ml. of 0.01 M aceticacid to obtain acid--soluble substances. To the extract was added 2 Mtrishydroxyamino-methane solution to adjust to pH 8.5, and the solutionwas then extracted three times with 100 ml. of chloroform. There wasobtained 230 mg. of red crude powder (baumycin complex) from thechloroform layer by concentration to dryness under reduced pressure.

EXAMPLE 2

The crude powder obtained as in Example 1 (230 mg.) was dissolved in 2ml. of chloroform-methanol mixture (10:1), subjected to a column 65 cm.in length and 8 cm. in diameter filled with 80 g. of silicic acid andwashed with chloroformmethanol mixture (10:1). The baumycin A₁ fractionwas eluted first, followed successively by baumycin A₂, B₁ and B₂ usingas the respective eluent 8:1, 5:1 and 2:1 mixtures ofchloroformmethanol.

After each active fraction was pooled separately and concentrated todryness under reduced pressure, each such fraction was applied to acolumn 25 cm. in length and 1.8 cm. in diameter filled with SephadexLH-20 and washed with 3:1 toluene-methanol mixture. After concentratingeach fraction obtained above, red powders of 10 mg. baumycin A₁, 18 mg.of baumycin A₂, 3 mg. baumycin B₁ and 1 mg. baumycin B₂ were obtained bythe addition of n-hexane to the concentrate.

EXAMPLE 3

A nutrient medium having the following composition was prepared:

    ______________________________________                                        Potato starch         2%                                                      Glucose               2%                                                      Yeast extract                                                                 (Daigo Eyo Co.)       0.5%                                                    NaCl                  0.25%                                                   Calcium carbonate     0.32%                                                   Soya meal (Nishin Oil KK)                                                                           2%                                                      Mineral*              0.2% (pH 7.4)                                           ______________________________________                                         *Mineral is same as in Example 1.                                        

Eight liters of the above medium were prepared of which 50 ml. each weredistributed in 500 ml.--flasks, sterilized at 120° C. for 15 min. andinoculated with 1 ml. of the seed culture of Streptomyces peuceticussubsp. carneus ATCC 21354 prepared by the method in Example 1.Fermentation was carried out on a rotary shaker at 28° C. for 6 days.The cultured broth was filtered to separate the mycelium from theculture filtrate. Extraction with chloroform and acetone proceeded as inExample 1, and 10 g. of oily substance was obtained. The oily substancewas dissolved in 100 ml. of methanol and 1.2 g. of precipitate wasobtained after removing n-hexane-soluble substances by addition of 100ml. of n-hexane. The red precipitate was dissolved in 100 ml. chloroformand extracted with 600 ml. of sodium acetate buffer (pH 3.0) to obtainan acid--soluble substance. After adding 0.5 Methylene-diaminetetraacetic acid to the extract to become 0.01 M, the pHwas adjusted to 8 with 4 M sodium hydrochloride.

The active components in this aqueous solution were extracted four timeswith 200 ml. of chloroform, and then extracted twice with 500 ml. ofn-butanol. The resulting chloroform- and n-butanol-layers wereconcentrated separately under reduced pressure, and 200 mg. of redpowder, which consisted mainly of baumycin B₂ was obtained. This crudepowder was dissolved in 5 ml. of chloroform-methanol, applied to acolumn 23 cm. in length and 3.0 cm. in diameter filled with 80 g. ofsilicic acid and washed with the 20:1 mixture of chloroform andmethanol. Baumycin B₁ and B₂ were eluted successively with the 5:1 and2:1 mixtures of chloroform and methanol. Fractions of baumycins B₁ andB₂ were concentrated separately under reduced pressure and 50 mg. ofbaumycin B₁ and 8 mg. of baumycin B₂ were obtained. The resultingbaumycin B₁ and B₂ were recrystallized from methanol, and 31 mg. and 6.5mg. of crystalline material were obtained, respectively. In this method,very little baumycin A₁ and A₂ are produced.

EXAMPLE 4

According to the general method of Examples 1 and 2, baumycin A₁, A₂, B₁and B₂ were obtained as follows using the indicated Streptomycesstrains:

    ______________________________________                                                          Baumycin obtained (mg.)                                     Strains             A.sub.1                                                                              A.sub.2                                                                              B.sub.1                                                                            B.sub.2                                ______________________________________                                        Streptomyces peuceticus subsp.                                                carneus ATCC 21354   8     12     4    2                                      Streptomyces coeruleorubidus                                                  ATCC 13740          16     21     8    3                                      Streptomyces peuceticus subsp.                                                caestus NRRL B-5337 11     10     7    3                                      Streptomyces peuceticus                                                       NRRL B-3826         10      5     1    3                                      Streptomyces coeruleorubidus                                                                      26     17     5    6                                      NRRL B-3045                                                                   ______________________________________                                    

We claim:
 1. A process for producing baumycin A₁ which comprisescultivating a baumycin A₁ -producing strain of Streptomyces selectedfrom the group consisting of Streptomyces coeruleorubidus ME 130-A4(FERM-P3540, ATCC 31276), Streptomyces peuceticus subsp. carneus ATCC21354, Streptomyces coeruleorubidus ATCC 13740, Streptomyces peuceticussubsp. caestus NRRL B-5337, Streptomyces peuceticus NRRL B-3826 andStreptomyces coeruleorubidus NRRL B-3045 in an aqueous nutrient mediumunder submerged aerobic conditions until a substantial amount ofbaumycin A₁ is produced by said organism in said culture medium andrecovering the baumycin A₁ from the culture medium substantially free ofsubstances co-produced therewith.
 2. A process for producing baumycin A₂which comprises cultivating a baumycin A₂ -producing strain ofStreptomyces selected from the group consisting of Streptomycescoeruleorubidus ME 130-A4 (FERM-P3540, ATCC 31276), Streptomycespeuceticus subsp. carneus ATCC 21354, Streptomyces coeruleorubidus ATCC13740, Streptomyces peuceticus subsp. caestus NRRL B-5337, Streptomycespeuceticus NRRL B-3826 and Streptomyces coeruleorubidus NRRL B-3045 inan aqueous nutrient medium under submerged aerobic conditions until asubstantial amount of baumycin A₂ is produced by said organism in saidculture medium and recovering the baumycin A₂ from the culture mediumsubstantially free of substances co-produced therewith.
 3. A process forproducing baumycin B₁ which comprises cultivating a baumycin B₁-producing strain of Streptomyces selected from the group consisting ofStreptomyces coeruleorubidus ME 130-A4 (FERM-P3540, ATCC 31276),Streptomyces peuceticus subsp. carneus ATCC 21354, Streptomycescoeruleorubidus ATCC 13740, Streptomyces peuceticus subsp. caestus NRRLB-5337, Streptomyces peuceticus NRRL B-3826 and Streptomycescoeruleorubidus NRRL B-3045 in an aqueous nutrient medium undersubmerged aerobic conditions until a substantial amount of baumycin B₁is produced by said organism in said culture medium and recovering thebaumycin B₁ from the culture medium substantially free of substancesco-produced therewith.
 4. A process for producing baumycin B₂ whichcomprises cultivating a baumycin B₂ -producing strain of Streptomycesselected from the group consisting Streptomyces coeruleorubidus ME130-A4 (FERM-P3540, ATCC 31276), Streptomyces peuceticus subsp. carneusATCC 21354, Streptomyces coeruleorubidus ATCC 13740, Streptomycespeuceticus subsp. caestus NRRL B-5337, Streptomyces peuceticus NRRLB-3826 and Streptomyces coeruleorubidus NRRL B-3045 in an aqueousnutrient medium under submerged aerobic conditions until a substantialamount of baumycin B₂ is produced by said organism in said culturemedium and recovering the baumycin B₂ from the culture mediumsubstantially free of substances co-produced therewith.